Imaging the rotational mobility of carbon dot-gold nanoparticle conjugates using frequency domain wide-field time-resolved fluorescence anisotropy

Abstract. Significance Wide-field measurements of time-resolved fluorescence anisotropy (TR-FA) provide pixel-by-pixel information about the rotational mobility of fluorophores, reflecting changes in the local microviscosity and other factors influencing the fluorophore’s diffusional motion. These features offer promising potential in many research fields, including cellular imaging and biochemical sensing, as demonstrated by previous works. Nevertheless, θ imaging is still rarely investigated in general and in carbon dots (CDs) in particular. Aim To extend existing frequency domain (FD) fluorescence lifetime (FLT) imaging microscopy (FLIM) to FD TR-FA imaging (TR-FAIM), which produces visual maps of the FLT and θ, together with the steady-state images of fluorescence intensity (FI) and FA (r). Approach The proof of concept of the combined FD FLIM/ FD TR-FAIM was validated on seven fluorescein solutions with increasing viscosities and was applied for comprehensive study of two types of CD-gold nano conjugates. Results The FLT of fluorescein samples was found to decrease from 4.01±0.01 to 3.56±0.02  ns, whereas both r and θ were significantly increased from 0.053±0.012 to 0.252±0.003 and 0.15±0.05 to 11.25±1.87  ns, respectively. In addition, the attachment of gold to the two CDs resulted in an increase in the FI due to metal-enhanced fluorescence. Moreover, it resulted in an increase of r from 0.100±0.011 to 0.150±0.013 and θ from 0.98±0.13 to 1.65±0.20  ns for the first CDs and from 0.280±0.008 to 0.310±0.004 and 5.55±1.08 to 7.95±0.97  ns for the second CDs. These trends are due to the size increase of the CDs-gold compared to CDs alone. The FLT presented relatively modest changes in CDs. Conclusions Through the combined FD FLIM/ FD TR-FAIM, a large variety of information can be probed (FI, FLT, r, and θ). Nevertheless, θ was the most beneficial, either by probing the spatial changes in viscosity or by evident variations in the peak and full width half maximum.

Prior to utilizing the polarized beam splitter, we conducted measurements on four fluoresceinglycerol solutions (containing 0%, 30%, 60%, and 80% glycerol) using a linear polarizer in the emission path oriented both parallel (0 0 /0 0 -excitation/emission) and perpendicular (0 0 /90 0 ) to the excitation vertical orientation. Upon investigating the impact of replacing the linear polarizer with a polarizing beam splitter, we noted that the manufacturer indicated a possible leakage of up to 5%. After repeating the measurements with the polarizing beam splitter, we observed a clear decrease in the r values. We then derived formulas to compensate for the leakage and discovered that a 5% leakage resulted in good agreement between the results obtained using a linear polarizer in the emission path and those obtained using the polarizing beam splitter.
The total corrected reflected component is described by Shifting the phase of each signal by exp  ⊥ , and using exp exp exp After setting ωt=0, we obtain ( )  is the molar absorptivity absorption spectra.
The Strickler-Berg formula is fundamental relation that applies to all fluorophores. However, this formula predicts the experimental FLT only for cases that follow several assumptions which are only often achieved in real systems. One of which is the absolute rigidity of the fluorophore in both the ground and excited state. An example to a rigid fluorophore is the fluorescein and thus, this equation can describe well the relation between the refractive index of the media and the experimental FLT for fluorescein. However, generally interactions between the fluorophore and its solvent mask this behavior (since different quenching effects frequently dominate) and Eq.

Appendix C
The histograms in the results section have normalized probability and they are characterized by the mode (the peak of each distribution) and the FWHM (the width of a distribution measured between the points on the y-axis which are half the maximum amplitude). For example, in Fig. S1, there are 3 different θ distributions of Fl-Gly system with 3 different glycerol concentration (30%, 50% and 80%). Clearly, the increase in glycerol concentration increases both the mode and the FWHM. Therefore, both the mode and the FWHM can imply on the increasing viscosity.

Appendix D
In order to confirm the attachment of each of the CDs to the AuNPs the morphologies and dimensions of the CDs and CDs-Au nanohybrid were investigated by TEM. The TEM images for the AuNPs alone found a size range from 14-18 nm ( Fig. S2(a)). In addition, a prominent peak in the CDs FTIR spectra (Fig. S2(b)) at 1515 cm -1 indicated the existence of the -NH group, whereas a broad peak at 1345 cm -1 indicated OH deformation vibrations. The change of the carbonyl peak of carboxylic acid from 1623 to 1609 cm -1 endorsing the establishment of an amide linkage confirmed the covalent attachment of AuNPs to the CD surface.

Appendix E
Prior to using the polarized beam splitter in the emission path, we conducted a series of measurements with a linear polarizer, rotating the orientation angle of the polarizer (α) by 10 degrees with each measurement. By doing so, we were able to obtain polarization intensity data that followed a COS 2 pattern between the orientation angle and intensity, thereby validating the exact angle of both the parallel and perpendicular components (Fig. S3).

Fig. S3.
The relation between the intensity and the orientation angle of the emission linear polarizer. A sequence of measurements was performed using a linear polarizer, where the orientation angle was altered by 10 degrees in each measurement. This approach enabled us to gather data on the polarization intensity that adhered to the expected COS 2 trend linking the angle of orientation to the intensity. Consequently, the precise angles for both the parallel and perpendicular components were confirmed.